Publications

Here is a selection of publications where different laminin isoforms are being used to create more authentic cell culture systems

  • Area of interest

  • The ability of inner-cell-mass cells to self-renew as embryonic stem cells is acquired following epiblast specification

    Boroviak T., Loos R., Bertone P., Smith A. and Nichols J.Nature Cell Biology, 2014

    The authors show that mouse ICM cells from early blastocysts can progress to ERK independence if provided with a specific laminin substrate. We show by RNA sequencing that Laminin-511 is expressed in the early ICM, as is B1-integrin, which mediates laminin binding in ESCs. No expression of Laminin-521 in mouse ICM. Single ESC derivation in 2i-LIF on a fibronectin-LN511 mix and clonal ESC lines expansion on LN511.

  • A Balance between Secreted Inhibitors and Edge Sensing Controls Gastruloid Self-Organization

    Etoc F., Metzger J., Ruzo A., Kirst C., Yoney A., Ozair Z.M, Brivanlou A.H., Siggia E.D. Developmental cell, 2016

    Used colonies of hESC grown on the micropatterned substrate and differentiated with BMP4 to model patterning events in the human embryo. Coated Costar Transwells with LN521.

  • CCR2+CCR5+ T Cells Producing Matrix Metalloproteinase-9 and Osteopontin in the Pathogenesis of Multiple Sclerosis

    Sato W., Tomita A., Ichikawa D., Lin Y., Kishida H., Miyake S., Ogawa M., Okamoto T., Murata M., Kuroiwa Y., Aranami T., Yamamura T.Journal of Immunology, 2012

    To recapitulate the glia limitans layered with parenchymal basal lamina experimentally, we coated the upper sides of Transwell membrane inserts. The upper sides of Transwell membrane inserts (8 mm; Corning) were coated with 10 mg/ml laminin-1 (Sigma) or 20 mg/ml laminin-121. After aspirating the laminin solutions, the membrane inserts were turned upside down, and normal human astrocytes (NHA) were seeded on the lower sides of the membrane inserts. T cells were stimulated, harvested, suspended in the fresh medium, and seeded onto the upper chambers. After 8 h, the cell suspension was collected from the lower chambers after careful pipetting, and absolute numbers of migrated cells were calculated. The T-cell migration across the NHA layered with laminin-111 or -121 was less efficient compared with the migration across the untreated membrane or the membrane treated with laminin alone, thus, this model would exhibit barrier functions against the penetration of activated T cells.

  • Early embryonic-like cells are induced by downregulating replication-dependent chromatin assembly

    Ishiuchi T., Enriquez-Gasca R., Mizutani E., Bošković A., Ziegler-Birling C., Rodriguez-Terrones D., Wakayama T., Vaquerizas J.M., Torres-Padilla M-E.Nature structural & molecular biology, 2015

    mES cells cultured on coverslips coated with laminin-511for an RNA-FISH assay.

  • Dynamic Heterogeneity and DNA Methylation in Embryonic Stem Cells

    Singer Z.S., Yong J., Tischler J., Hackett J.A., Altinok A., Surani M.A., Cai L., and Elowitz M.B.Cell Press, 2014

    Analyzis of gene expression in individual ESCs using single-molecule RNA-FISH and quantitative time-lapse movies. Fluorescence Time-Lapse Microscopy and Data analysis on reporter cells mixed with unlabeled parental cells at 1:9 ratio and plated at a total density of 20,000 cells/cm2 on glass-bottom plates (MatTek) coated with human laminin-511 >12 hr before imaging.

  • Control of ground-state pluripotency by allelic regulation of Nanog

    Miyanari Y., Torres-Padilla M.E.Nature Letter, 2012

    Laminin-511 used to coat glass-bottomed dishes.

  • Live visualization of chromatin dynamics with fluorescent TALEs

    Miyanari Y., Ziegler-Birling C., Torres-Padilla M.E.Nature structural & molecular biology, 2013

    The authors of these two Nature publications show that laminin can be coated directly on glass, which many other substrates and proteins can not. This enables the growth of pluripotent stem cells as monolayers even on glass, which is especially suitable for live imaging.

  • Improved Human Pluripotent Stem Cell Attachment and Spreading on Xeno-Free Laminin-521-Coated Microcarriers Results in Efficient Growth in Agitated Cultures

    Alan Tin-Lun Lam, Jian Li, Allen Kuan-Liang Chen, William R Birch, Shaul Reuveny, Steve Kah-Weng Oh. Biores Open Access, 2015

    The authors used different bioreactors up to 2.5 L in scale, and successfully cultured hMSCs using the minimum agitator speed required for complete microcarrier suspension. In addition, we also reported a scaleable protocol for the detachment from microcarriers in spinner flasks where they use a short period of intense agitation in the presence of enzymes such that the cells are detached without being damaged. Here, the same approach has been effective for 15 mL ambrTMbioreactors, 100 mL spinner flasks and 250 mL Dasgip bioreactors.  Two types of microcarrier were used, with (laminin-521) and without surface coatings, four different enzymes and three different growth media (with and without serum), a total of 22 different combinations. In all cases after detachment, the cells were shown to retain their desired quality attributes and were able to proliferate. Qasim Rafiq presented this data at BioLamina symposium 2015. It was found that human recombinant laminin LN-521 and recombinant fibronectin were the optimal ECM proteins for microcarrier coating. A serum-free expansion, harvest and xeno, and DMSO-free cryopreservation process, using LN-521 coated microcarriers and FREEZEstemTM cryopreservation medium was then developed which demonstrated > 5 fold increase in hMSC yield.

  • Collaboration of 3D Context and Extracellular Matrix in the Development of Glioma Stemness in a 3D Model

    Ma N.K.L., Kai Lim J., Fatt Leong M., Sandanaraj E., Ti Ang B., Tang C., Wan A.C.A. Biomaterials, 2015

    This work illustrates that different laminin isoforms have specific effects in promoting the stemness of glioma cells, in collaboration with a 3D context. U251 glioblastoma cells were cultured on electrospun polystyrene (ESPS) scaffolds coated with 7 different laminin isoforms (LAMscreen kit) to provide a 3D model for stem cell-related genes and proteins expression studies. The results indicate the influence of 3D (versus 2D) context on stemness markers and integrin expression, specifically, the upregulation of the laminin-binding integrins a6b4. By a colony-forming assay, we showed enhanced clonogenicity of cells grown on ESPS scaffolds in collaboration with laminins 411, 421, 511 and 521. The present results demonstrate how 3D versus 2D context profoundly affects ECM signaling, leading to stemness.

  • Enhanced reseeding of decellularized rodent lungs with mouse embryonic stem cells

    Lecht S., Stabler C.T., Rylander A.L., Chiaverelli R., Schulman E.S., Marcinkiewicz C., Lelkes P.I.Biomaterials, 2014

    Pre-treatment of the decellularized lung with defined ECM proteins, to evaluate the efficacy of reseeding of mESC. All current decellularization methodologies result in significant alterations of the contents, and ratios of ECM proteins, though the exact degree of the loss of ECM glycoproteins, such as laminins. Following ECMs were tested: bovine collagen type IV; human collagen type IV; human pro-collagen type I; human collagen type I; rat collagen type I; human plasma-purified fibronectin; human thrombospondin-1; VCAM; vitronectin; bovine elastin; Matrigel-purified laminin 111; and human recombinant laminins 111, 211, 332, 411, 421, 511, 521. mESCs lack major integrin receptors for collagens but express high levels of functional receptors for LM and FN. Laminin next to FN appears to be the major pro-adhesive ECM protein for mESCs. The mESCs were found to adhere differentially in an isoform-dependent manner with the following order of potencies 511 = 521 >332 >421 >211 >111 >411. These findings further support the notion that a3b1 and a6b1 integrin receptors expressed on the mES cell surface are involved in the specific binding to LM in general and to LM 511 and 521. LM and FN interact more efficiently with collagen type I than with collagen IV or elastin 90% of the available Coll I in the decellularized lung matrix forms a complex with LM. in vitro that cell-derived FN and LM interact with Coll I with much higher efficiencies than commercial FN or LM This finding may be explained by a possible partial loss of activity as a result of the purification procedures in contrast to the unprocessed CM.